Arylheteroalkylamine derivatives and their use as inhibitors of nitric oxide synthase

ABSTRACT

There are provided novel compounds of formula (I), wherein R 1 , R 2 , R 3 , T, U, X, Y, V and W are as defined in the specification, and pharmaceutically acceptable salts thereof, and enantiomers and racemates thereof; together with processes for their preparation, compositions containing them and their use in therapy. The compounds are inhibitors of nitric oxide synthase and are thereby particularly useful in the treatment or prophylaxis of inflammatory disease and pain

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the national phase application under 35 U.S.C. § 371of PCT International Application No. PCT/SE02/01414, which has anInternational filing date of Jul. 26, 2002, and which designated SwedishApplication Serial No. 0102640-0, filed Jul. 31, 2001, as priority. Thecontents of these applications are incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to novel arylheteroalkylamine derivatives,processes for their preparation, compositions containing them and theiruse in therapy.

BACKGROUND OF THE INVENTION

Nitric oxide is produced in mammalian cells from L-arginine by theaction of specific nitric oxide synthases (NOSs). These enzymes fallinto two distinct classes—constitutive NOS (cNOS) and inducible NOS(iNOS). At the present time, two constitutive NOSs and one inducible NOShave been identified. Of the constitutive NOSs, an endothelial enzyme(eNOS) is involved with smooth muscle relaxation and the regulation ofblood pressure and blood flow, whereas the neuronal enzyme (nNOS)appears to be involved in the regulation of various biologicalfunctions. Inducible NOS has been particularly implicated in thepathogenesis of inflammatory diseases. Regulation of these enzymesshould therefore offer considerable potential in the treatment of a widevariety of disease states (J. E. Macdonald, Ann. Rep. Med. Chem., 1996,31, 221–230).

Considerable effort has been expended to identify compounds that act asspecific inhibitors of one or more isoforms of the enzyme nitric oxidesynthase. The use of such compounds in therapy has also been widelyclaimed.

DISCLOSURE OF THE INVENTION

According to the present invention, there is provided a compound offormula (I)

wherein:

-   X represents H, C1 to 4 alkyl, C1 to 4 alkoxy, halogen, OH, CN,    C≡CH, NO₂, CHO, COCH₃ or NHCHO; said alkyl or alkoxy group being    optionally further substituted by one or more fluorine atoms or by    an OH group;-   Y represents C1 to 4 alkyl, C1 to 4 alkoxy, halogen, OH, CN, CH,    NO₂, CHO, COCH₃ or NHCHO; said alkyl or alkoxy group being    optionally further substituted by one or more fluorine atoms;-   Either one of T, U and W represents N and the other two    independently represent CR⁴; or each of T, U and W represents CR⁴;    and each R⁴ group independently represents H, F or CH₃;-   V represents O or S(O)_(n);-   n represents an integer 0, 1 or 2;-   R¹ represents C1 to 4 alkyl, C2 to 4 alkenyl, C2 to 4 alhynyl, C3 to    6 cycloalkyl or a 4 to 8 membered saturated heterocyclic ring    incorporating one heteroatom selected from O, S and N; any of said    groups being optionally further substituted by C1 to 4 alkyl, C1 to    4 alkoxy, C1 to 4 alkylthio, C3 to 6 cycloalkyl, one or more    halogens or phenyl; said phenyl group being optionally further    substituted by one or more substituents selected independently from    halogen, C1 to 4 alkyl, C1 to 4 alkoxy, CF₃, OCF₃, CN or NO₂;-   or R¹ represents phenyl or a five or six membered aromatic    heterocyclic ring containing 1 to 3 heteroatoms independently    selected from O, S and N; said phenyl or aromatic heterocyclic ring    being optionally substituted by one or more substituents selected    independently from halogen, C1 to 4 alkyl, C1 to 4 alkoxy, OH, CN,    NO₂ or NR⁵R⁶; said alkyl or alkoxy group being optionally further    substituted by one or more fluorine atoms;-   R² and R³ independently represent H, C1 to 4 alkyl or C3 to 6    cycloalkyl; said alkyl group being optionally substituted by one or    more groups selected independently from C1 to 4 alkoxy, halogen, OH,    NR⁷R⁸, ═NR⁷, phenyl or a five or six membered aromatic or saturated    heterocyclic ring containing 1 to 3 heteroatoms independently    selected from O, S and N; said phenyl or aromatic heterocyclic ring    being optionally further substituted by halogen, C1 to 4 alkyl, C1    to 4 alkoxy, CF₃, OCF₃, CN or NO₂.

R⁵, R⁶, R⁷ and R⁸ independently represent H or C1 to 4 alkyl;

or a pharmaceutically acceptable salt thereof.

It will be recognised that compounds of formula (I) wherein W representsN and X represents OH may exist in the alternative tautomeric form (Ia):

Analogous tautomeric structures will also exist for compounds of formula(I) wherein T represents N and X represents OH; or wherein U representsN and Y represents OH.

Compounds of formula (I) wherein R² represents H and R³ represents C1 to4 alkyl substituted on the α-carbon atom by ═NR⁷ may also exist intautomeric forms such as those shown in formula (Ib):

All possible tautomers of compounds of formula (I) and mixtures thereofare included within the scope of the present invention.

The compounds of formula I may exist in enantiomeric forms. Allenantiomers, diastereomers, racemates and mixtures thereof are includedwithin the scope of the invention.

The compounds of formula (I) and their pharmaceutically acceptable saltshave the advantage that they are inhibitors of the enzyme nitric oxidesynthase (NOS). In general, the compounds of formula (I) and theirpharmaceutically acceptable salts have the advantage that they areinhibitors of the inducible isoform of the enzyme nitric oxide synthase(iNOS). Certain compounds of formula (I) and their pharmaceuticallyacceptable salts have the advantage that they are additionally oralternatively inhibitors of the neuronal isoform of the enzyme nitricoxide synthase (nNOS). In general, compounds of formula (I) and theirpharmaceutically acceptable salts have the advantage that they show goodselectivity for the inhibition of INOS and/or nNOS in comparison to theinhibition of the endothelial isoform, eNOS.

The invention further provides a process for the preparation ofcompounds of formula (I) or a pharmaceutically acceptable salt,enantiomer or racemate thereof.

According to the invention there is also provided a compound of formula(I), or a pharmaceutically acceptable salt thereof, for use as amedicament.

Another aspect of the invention provides the use of a compound offormula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatmentor prophylaxis of humandiseases or conditions in which inhibition of nitric oxide synthaseactivity is beneficial.

A more particular aspect of the invention provides the use of a compoundof formula (I) or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament, for the treatment or prophylaxis ofinflammatory disease.

Another more particular aspect of the invention provides the use of acompound of formula (I) or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament, for is the treatment or prophylaxisof CNS disease.

According to the invention, there is also provided a method of treating,or reducing the risk of, diseases or conditions in which inhibition ofnitric oxide synthase activity is beneficial which comprisesadministering to a person suffering from or at risk of, said disease orcondition, a therapeutically effective amount of a compound of formula(I) or a pharmaceutically acceptable salt thereof.

More particularly, there is also provided a method of treating, orreducing the risk of, inflammatory disease in a person suffering from orat risk of, said disease, wherein the method comprises administering tothe person a therapeutically effective amount of a compound of formula(I) or a pharmaceutically acceptable salt thereof.

More particularly, there is also provided a method of treating, orreducing the risk of, CNS disease in a person suffering from or at riskof, said disease, wherein the method comprises administering to theperson a therapeutically effective amount of a compound of formula (I)or a pharmaceutically acceptable salt thereof.

The compounds of the present invention may also be used advantageouslyin combination with a second pharmaceutically active substance;particularly in combination with a cyclooxygenase inhibitor; moreparticularly in combination with a selective inhibitor of the inducibleisoform of cyclooxygenase (COX-2). Thus, in a further aspect of theinvention there is provided the use of a compound of formula (I) or apharmaceutically acceptable salt thereof, in combination with a COX-2inhibitor in the manufacture of a medicament for the treatment ofinflammation, inflammatory disease and inflammatory related disorders.And there is also provided a method of treating, or reducing the riskof, inflammation, inflammatory disease and inflammatory relateddisorders in a person suffering from or at risk of, said disease orcondition, wherein the method comprises administering to the person atherapeutically effective amount of a compound of formula (I) or apharmaceutically acceptable salt thereof in combination with a COX-2inhibitor.

In one embodiment, R² and R³ independently represent H, C1 to 4 alkyl orC3 to 6 cycloalkyl; said alkyl group being optionally substituted by C1to 4 alkoxy, halogen, OH, NR⁷R⁸, phenyl or a five or six memberedaromatic or saturated heterocyclic ring containing 1 to 3 heteroatomsindependently selected from O, S and N; said phenyl or aromaticheterocyclic ring being optionally further substituted by halogen, C1 to4 alkyl, C1 to 4 alkoxy, CF₃, OCF₃, CN or NO₂; and X, Y, T, U, W, V, n,R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are as defined above.

In one embodiment, V represents S(O)_(n) and n represents 0.

In another embodiment, V represents O.

In another embodiment, X and Y independently represent Br, Cl, CH₃,CH₂F, CHF₂, CF₃ or CN. In yet another embodiment Y represents CN.

In another embodiment, R¹ represents phenyl, pyridyl, thienyl,isoxazolyl, isothiazolyl or thiazolyl. In a further embodiment, R¹represents phenyl.

In one embodiment, R² represents H or CH₃.

In one embodiment, each R³ represents H or CH₃.

In another embodiment, T, U and W each represent CH or CF.

In another embodiment, one of the groups T, U and W represents N, andthe other two groups independently represent CH or CF. In a particularembodiment W represents N and T and U each represent CH.

In a particular embodiment, the compounds of formula (I) have theabsolute stereochemistry as shown in formula (Ic):

In one particular aspect the invention relates to compounds of formula(I) wherein V represents O or S; X and Y independently represent Br, Cl,CH₃, CH₂F, CHF₂, CF₃ or CN; R¹ represents phenyl, pyridyl, thienyl,isoxazolyl, isothiazolyl or thiazolyl; R² and R³ each represent H orCH₃; T and U each represent CH or CF; W represents CH or N; and thecompounds have the absolute configuration shown in formula (Ic); andpharmaceutically acceptable salts thereof.

Particular compounds of the invention include:

-   2-{[(1S)-2-amino-1-phenylethyl]oxy}-4-chloro-5-fluorobenzonitrile;-   2-[[(1S)-2-amino-1-phenylethyl]thio]-6-methyl-3-pyridinecarbonitrile;-   2-[(2,5-dichlorophenyl)thio]-2-phenylethylamine;-   2-[[(1S)-2-amino-1-phenylethyl]thio]-4-chlorobenzonitrile;-   N-[2-(5-chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-ethanimidamide;-   N-[2-(5-chloro-2-cyano-4-Auorophenoxy)-2-phenylethyl]-2-hydroxyethanimidade;-   2-[[(1S)-2-amino-1-phenylethyl]thio]-6-(trifluoromethyl)-3-pyridinecarbonitrile;-   2-[[(1S)-2-amino-1-phenylethyl]thio]-4-methoxybenzonitrile;-   4-chloro-2-[[(1S)-2-(methylamino)-1-phenylethyl]thio]benzonitrile;-   2-[[(1R)-2-amino-1-(3-isoxazolyl)ethyl]oxy]-4-chloro-5-fluorobenzonitrile;-   2-[[2-amino-1-(3-pyridinyl) ethyl]thio]-4-chlorobenzonitrile;-   and pharmaceutically acceptable salts thereof.

Unless otherwise indicated, the term “C1 to 4 alkyl” referred to hereindenotes a straight or branched chain alkyl group having from 1 to 4carbon atoms. Examples of such groups include methyl, ethyl, n-propyl,i-propyl, n-butyl, i-butyl and t-butyl.

Unless otherwise indicated, the term “C3 to 6 cycloalkyl” referred toherein denotes a cycloalkyl group having from 3 to 6 carbon atoms.Examples of such groups include cyclopropyl, cyclopentyl and cyclohexyl.

Unless otherwise indicated, the term “C2 to 4 alkenyl” referred toherein denotes a straight or branched chain alkyl group having from 2 to4 carbon atoms incorporating at least one carbon-carbon double bond.Examples of such groups include ethenyl, propenyl and butenyl.

Unless otherwise indicated, the term “C2 to 4 alkynyl” referred toherein denotes a straight or branched chain alkyl group having from 2 to4 carbon atoms incorporating at least one carbon-carbon triple bond.Examples of such groups include ethynyl, propynyl, and butynyl.

Unless otherwise indicated, the term “C1 to 4 alkoxy” referred to hereindenotes a straight or branched chain alkoxy group having from 1 to 4carbon atoms. Examples of such groups include methoxy, ethoxy,n-propoxy, i-propoxy and t-butoxy.

The term “C1 to 4 alkylthio” is to be interpreted analogously.

Unless otherwise indicated, the term “halogen” referred to hereindenotes fluoro, chloro, bromo and iodo.

Examples of a 4 to 8 membered saturated heterocyclic ring incorporatingone heteroatom selected from O, S or N include pyrrolidine, piperidine,tetrahydrofuran and perhydroazepine.

Examples of a five or six membered aromatic heterocyclic ring containing1 to 3 heteroatoms independently selected from O, S and N include furan,thiophene, pyridine, thiazole, imidazole, oxazole, triazole, oxadiazole,thiadiazole and pyrimidine.

Examples of a five or six membered saturated heterocyclic ringcontaining 1 to 3 heteroatoms independently selected from O, S and Ninclude morpholine, pyrrolidine, tetrahydrofuran, piperidine andpiperazine.

Examples of a “C1 to 4 alkyl or C1 to 4 alkoxy optionally furthersubstitutedby one or more fluorine atoms” include CH₂F, CHF₂, CF₃,CF₃CF₂, CF₃CH₂, CH₂FCH₂, CH₃CF₂, CF₃CH₂CH₂, OCF₃ and OCH₂CF₃.

According to the invention, we further provide a process for thepreparation of compounds of formula (I), or a pharmaceuticallyacceptable salt, enantiomer or racemate thereof which comprises:

-   (a) reaction of a compound of formula (II)

wherein T, U, X, Y and W are as defined in formula (I) and L¹ representsa leaving group, with a compound of formula (III)

wherein R¹, R², R³ and V are as defined in formula (I); or

-   (b) reaction of a compound of formula (IV)

wherein T, U, W, X, Y and V are as defined in formula (I), with acompound of formula (V)

wherein R¹, R² and R³ are as defined in formula (I) and L² is a leavinggroup; or

-   (c) reaction of a compound of formula (VI)

wherein R¹, T, U, W, X, Y and V are as defined in formula (I) and L³ isa leaving group, with a compound of formula (VII)R²R³NH  (VII)wherein R², and R³ are as defined in formula (I); or

-   (d) reduction of a compound of formula (VIII)

wherein R¹, T, U, W, X, Y and V are as defined in formula (I) and Qrepresents azide (N₃); or

-   (e) hydrolysis of a compound of formula (VIII)

wherein R¹, T, U, W, X, Y and V are as defined in formula (I) and Qrepresents an imide group;and where desired or necessary converting the resultant compound offormula (I), or another salt thereof, into a pharmaceutically acceptablesalt thereof; or converting one compound of formula (I) into anothercompound of formula (I); and where desired converting the resultantcompound of formula (I) into an optical isomer thereof.

In process (a), the reaction is performed by treating a nucleophile offormula (III) with an electrophile of formula (II) in an inert solvent.Suitable leaving groups L¹ include sulphonates and halides, particularlyfluoride or chloride. The reaction is generally performed in thepresence of a non-nucleophilic base such as sodium hydride or caesiumcarbonate. Suitable organic solvents are those such asN,N-dimethylformamide, N-methyl-2-pyrrolidinone, tetrahydrofuran anddimethylsulfoxide. The reaction is generally conducted at a temperaturebetween 0° C. and the boiling point of the solvent.

In process (b), the reactants (IV) and (V) are coupled together in asuitable inert solvent such as tetrahydrofuran using, for example,Mitsunobu conditions. Thus, for example, the reactants are treated witha phosphine derivative and an azo derivative at a suitable temperature,generally between 0° C. and the boiling point of the solvent. Suitablephosphine derivatives include triphenylphosphine and tributylphosphine.Suitable azo derivatives include diethyl azodicarboxylate, diisopropylazodicarboxylate and 1,1′-(azodicarbonyl)dipiperidine. Suitable leavinggroups L² include hydroxy.

Alternatively in process (b), the reaction is performed by treating anucleophile of formula (IV) with an electrophile of formula (V) in aninert solvent. Suitable leaving groups L² include sulphonates andhalides, particularly chloride or bromide. The reaction is generallyperformed in the presence of a non-nucleophilic base such as sodiumhydride or caesium carbonate. Suitable organic solvents are those suchas N,N-dimethylformamide, N-methyl-2-pyrrolidinone, tetrahydrofuran anddimethylsulfoxide. The reaction is generally conducted at a temperaturebetween 0° C. and the boiling point of the solvent.

In process (c), the compounds of formulae (VI) and (VII) are reactedtogether in a suitable inert solvent such as dimethylsulphoxide ortetrahydrofuran. The reaction is generally carried out in the presenceof a base. The base may be either an added component or an excess of theamine (VII). Suitable leaving groups L³ include iodide andp-toluenesulphonate.

In processes (d) and (e), the reactions are carried out using standardconditions that will be well known to the man skilled in the art.

It will be apparent to a person skilled in the art that in the aboveprocesses it may be desirable or necessary to protect an amine orhydroxyl or other potentially reactive group.

Suitable protecting groups and details of processes for adding andremoving such groups may be found by reference to the standard text“Protective Groups in Organic Synthesis”, 3rd Edition (1999) by Greeneand Wuts.

In one preferred embodiment, amine groups are protected as carbamatederivatives, for example, as t-butyloxycarbamates.

Specific examples of the use of protecting groups are given in theExamples section.

The present invention includes compounds of formula (I) in the form ofsalts, in particular acid addition salts. Suitable salts include thoseformed with both organic and inorganic acids. Such acid addition saltswill normally be pharmaceutically acceptable although salts ofnon-pharmaceutically acceptable acids may be of utility in thepreparation and purification of the compound in question. Thus,preferred salts include those formed from hydrochloric, hydrobromic,sulphuric, phosphoric, citric, tartaric, lactic, pyruvic, acetic,succinic, fumaric, maleic, methanesulphonic and benzenesulphonic acids.

Salts of compounds of formula (I) may be formed by reacting the freebase, or a salt, enantiomer or racemate thereof, with one or moreequivalents of the appropriate acid. The reaction may be carried out ina solvent or medium in which the salt is insoluble or in a solvent inwhich the salt is soluble, for example, water, dioxane, ethanol,tetrahydrofuran or diethyl ether, or a mixture of solvents, which may beremoved in vacuo or by freeze drying.

The reaction may also be a metathetical process or it may be carried outon an ion exchange resin.

Compounds of formula (III) may be prepared by reaction of a compound offormula (IX)

wherein R² and R³ are as defined in formula (I), and G represents H, Clor NCH₃(OCH₃), with an organometallic derivative, R¹-M, wherein R¹ is asdefined in formula (I) and M represents a metallic residue such aslithium or magnesium-halide, followed if necessary by reduction. Theresulting compound of formula (III) wherein V represents oxygen may thenbe subsequently converted into compounds of formula (III) wherein Vrepresents sulphur.

Compounds of formulae (II), (IV), (V), (VI), (VIII) and (IX) are eitherknown or may be prepared by conventional methods that will be readilyapparent to the man skilled in the art.

Intermediate compounds may be used as such or in protected form.Protecting groups and details of processes for their removal may befound by reference to the standard text “Protective Groups in OrganicSynthesis”, 3rd Edition (1999) by Greene and Wuts.

The compounds of the invention and intermediates thereto may be isolatedfrom their reaction mixtures and, if necessary further purified, byusing standard techniques.

The compounds of formula I may exist in enantiomeric forms. Therefore,all enantiomers, diastereomers, racemates and mixtures thereof areincluded within the scope of the invention. The various optical isomersmay be isolated by separation of a racemic mixture of the compoundsusing conventional techniques, for example, fractional crystallisation,or HPLC.

Intermediate compounds may also exist in enantiomeric forms and may beused as purified enantiomers, diastereomers, racemates or mixtures.

The compounds of formula (I), and their pharmaceutically acceptablesalts are useful because they possess pharmacological activity inanimals. In particular, the compounds are active as inhibitors of theenzyme nitric oxide synthase. More particularly, they are inhibitors ofthe inducible isoform of the enzyme nitric oxide synthase and as suchare predicted to be useful in therapy, for example, as anti-inflammatoryagents. Alternatively, they may have utility as inhibitors of theneuronal isoform of the enzyme nitric oxide synthase and as such arepredicted to have utility in the treatment of CNS disorders.

The compounds and their pharmaceutically acceptable salts are indicatedfor use in the treatment or prophylaxis of diseases or conditions inwhich synthesis or oversynthesis of nitric oxide forms a contributorypart. In one aspect, the compounds are indicated for use in thetreatment of inflammatory conditions in mammals including man. Inanother aspect, the compounds are indicated for use in the treatment ofCNS disorders in mammals including man.

Conditions that may be specifically mentioned are:

-   osteoarthitis, rheumatoid arthritis, rheumatoid spondylitis, gouty    arthritis and other arritic conditions, inflamed joints;-   eczema, psoriasis, dermatitis or other inflammnatory skin conditions    such as sunburn;-   inflammatory eye conditions including uveitis, glaucoma and    conjunctivitis;-   lung disorders in which inflammation is involved, for example,    astbina, bronchitis, chronic obstructive pulmonary disease, pigeon    fancier's disease, farmer's lung, acute respiratory distress    syndrome;-   bacteraeinia, endotoxaemia (septic shock), aphthous ulcers,    gingivitis, pyresis, pain, meningitis and pancreatitis;-   conditions of the gastrointestinal tract including inflammatory    bowel disease, Crohn's disease, atrophic gastritis, gastritis    varialoforme, ulcerative colitis, coeliac disease, regional ileitis,    peptic ulceration, irritable bowel syndrome, reflux oesophagitis,    damage to the gastrointestinal tract resulting from infections by,    for example, Helicobacter pylori, or from treatments with    non-steroidal anti-inflammatory drugs;    and other conditions associated with inflammation.

The compounds will also be useful in the treatment and alleviation ofacute pain or persistent inflammatory pain or neuropathic pain or painof a central origin.

The compounds may also be useful in the treatment of cancer.

We are particularly interested in the conditions inflammatory boweldisease, rheumatoid arthritis, osteoarthritis, chronic obstructivepulmonary disease, cancer and pain.

The compounds of formula (I) and their pharmaceutically acceptable saltsmay also be useful in the treatment or prophylaxis of diseases orconditions in addition to those mentioned above. For example, thecompounds may be useful in the treatment of atherosclerosis, cysticfibrosis, hypotension associated with septic and/or toxic shock, in thetreatment of dysfunction of the immune system, as an adjuvant toshort-term immunosuppression in organ transplant therapy, in the controlof onset of diabetes, in the maintenance of pancreatic function indiabetes, in the treatment of vascular complications associated withdiabetes and in co-therapy with cytokines, for example TNF orinterleukins.

The compounds of formula (I) may alternatively be useful in thetreatment of hypoxia, for example in cases of cardiac arrest and stroke,neurodegenerative disorders including nerve degeneration and/or nervenecrosis in disorders such as ischaeinia, hypoxia, hypoglycaemia,epilepsy, and in external wounds (such as spinal cord and head injury),hyperbaric oxygen convulsions and toxicity, dementia, for examplepre-senile dementia, Alzheimer's disease and AIDS-related dementia,Sydenham's chorea, Parkinson's disease, Tourette's Syndrome,Huntington's disease, Amyotrophic Lateral Sclerosis, Multiple Sclerosis,muscular dystrophy, Korsakoff s disease, imbecility relating to acerebral vessel disorder, sleeping disorders, schizophrenia, depression,pain, autism, seasonal affective disorder, jet-lag, depression or othersymptoms associated with Premenstrual Syndrome (PMS), anxiety and septicshock.

Compounds of formula (I) may also be expected to show activity in theprevention and reversal of drug addiction or tolerance such as toleranceto opiates and diazepines, treatment of drug addiction, treatment ofmigraine and other vascular headaches, neurogenic inflammation, in thetreatment of gastrointestinal motility disorders and in the induction oflabour.

We are particularly interested in the conditions stroke, Alzheimer'sdisease, Parkinson's disease, multiple sclerosis, schizophrenia,migraine, septic shock and pain; more particularly migraine.

Prophylaxis is expected to be particularly relevant to the treatment ofpersons who have suffered a previous episode of, or are otherwiseconsidered to be at increased risk of, the disease or condition inquestion. Persons at risk of developing a particular disease orcondition generally include those having a family history of the diseaseor condition, or those who have been identified by genetic testing orscreening to be particularly susceptible to developing the disease orcondition.

For the above mentioned therapeutic indications, the dosage administeredwill, of course, vary with the compound employed, the mode ofadministration and the treatment desired. However, in general,satisfactory results are obtained when the compounds are administered ata dosage of the solid form of between 1 mg and 2000 mg per day.

The compounds of formula (I), and pharmaceutically acceptablederivatives thereof, may be used on their own, or in the form ofappropriate pharmaceutical compositions in which the compound orderivative is in admixture with a pharmaceutically acceptable adjuvant,diluent or carrier. A further aspect of the invention provides apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier. Administrationmay be by, but is not limited to, enteral (including oral, sublingual orrectal), intranasal, inhalation, intravenous, topical or otherparenteral routes. Conventional procedures for the selection andpreparation of suitable pharmaceutical formulations are described in,for example, “Pharmaceuticals—The Science of Dosage Form Designs”, M. E.Aulton, Churchill Livingstone, 1988. The pharmaceutical compositionpreferably comprises less than 80% and more preferably less than 50% ofa compound of formula (I), or a pharmaceutically acceptable saltthereof.

There is also provided a process for the preparation of such apharmaceutical composition which comprises mixing the ingredients.

The compounds of formula (I), and pharmaceutically acceptablederivatives thereof, may also be advantageously used in combination witha COX inhibitor, more particularly in combination with a COX-2inhibitor. Particularly preferred COX-2 inhibitors are Celecoxib andMK-966. The NOS inhibitor and the COX-2 inhibitor may either beformulated together within the same pharmaceutical composition foradministration in a single dosage unit, or each component may beindividually formulated such that separate dosages may be administeredeither simultaneously or sequentially.

The invention is illustrated, but in no way limited, by the followingexamples:

EXAMPLE 12-[[(1S)-2-Amino-1-phenylethyl]oxy]-4-chloro-5-fluorobenzonitrile(E)-butenedioate

a) (S)-α-(Azidomethyl)benzenemethanol

(S)-α-(Chloromethyl)benzenemethanol (2.38 g) in dry DMSO (50 ml) wastreated with sodium azide (1.48 g) with stirring then heated at 60° C.for 24 h. More sodium azide (74 mg) was added and heating continued at80° C. for a further 24 h. The reaction mixture was cooled, poured intowater and extracted into ethyl acetate, which was washed with water (3×)then brine and dried (MgSO₄). The solvent was evaporated to give thesub-title compound (2.40 g) as a yellow oil.

¹H NMR 300 MHz (CDCl₃) 7.34–7.24 (5H, m), 4.83 (1H, m), 3.40 (2H, m).

b) 2-[[(1S)-2-Azido-1-phenylethyl]oxy]-4-chloro-5-fluorobenzonitrile

The product from step (a) (1.75 g) and 4-chloro-2,5-difluorobenzonitrile(1.86 g) in dry DMF (40 ml) were treated with 60% sodium hydride inmineral oil (470 mg) with stirring under nitrogen. The reaction mixturewas stirred for 2.5 h, poured into water, and extracted with ethylacetate. The extract was washed with water (5×) then brine and dried(MgSO₄). The solvent was evaporated and the residue purified bychromatography (silica, 10% ether/isohexane as eluent) to give thesub-title compound (2.37 g) as a pale yellow oil.

¹H NMR 300 MHz (CDCl₃) 7.39–7.26 (6H, m), 6.8 (1H, d), 5.24 (1H, m),3.79 (1H, m), 3.40 (1H, dd).

c) 2-[[(1S)-2-Amino-1-phenylethyl]oxy]-4-chloro-5-fluorobenzonitrile(E)-butenedioate

The product from step (b) (2.79 g) in dry THF (80 ml) was treated withtriphenylphosphine (3.47 g) and water (12 ml) and the mixture heated at60° C. for 2 h. The reaction mixture was cooled, poured into water, andextracted with ethyl acetate which was washed with water and dried(MgSO₄). The solvent was evaporated and the residue purified bychromatography (silica, ethyl acetate and then 10% 7M ammonia inmethanol/dichloromethane as eluent) to give an oil. This was dissolvedin ethanol (10 ml), treated with fumaric acid (70.3 mg) and heated untila clear solution was obtained. The mixture was evaporated and theresidue triturated with ether to give after filtration and drying invacuo the final compound (222 mg).

MS APCI+ve^(m)/z 291 ([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 8.0 (1H, d),7.54 (1H, d), 7.47–7.31 (5H, m), 6.52 (2H, s), 5.75 (1H, m), 3.21 (1H,m), 3.08 (1H, dd).

EXAMPLE 22-[[(1S)-2-Amino-1-phenylethyl]thio]-6-methyl-3-pyridinecarbonitrilehydrochloride

a) 1,1-Dimethylethyl[(2R)-2-hydroxy-2-phenylethyl]carbamate

(R)-α-(ainiomethyl)benzenemethanol (1.70 g) andbis(1,1-deimethylethyl)carbonate (2.98 g) were dissolved in methanol (20ml) and triethylamine (4.3 ml) added. The mixture was stirred at roomtemperature for 4 days and then concentrated to leave the sub-title iscompound as an oil (2.60 g, 88%).

¹H NMR 300 MHz (CDCl₃) 7.27–7.38 (5H, m), 4.92 (1H, m), 4.83 (1H, m),3.50 (1H, m), 3.25 (1H, m), 1.45 (9H, s).

b)S-[(1S)-2-[[(1,1-Dimethylethoxy)carbonyl]ainino]-1-phenylethyl]benzenecarbothioate

To a solution of triphenylphosphine (5.75 g) in THF (100 ml) undernitrogen at 0° C. was added diisopropylazodicarboxylate (4.55 ml)dropwise. The mixture was stirred at 0° C. for 45 minutes and then asolution of thiobenzoic acid (3.05 ml) and the product from step (a)(2.60 g) in THF (50 ml) was added dropwise at 0° C. After the additionwas complete the mixture was stirred at room temperature for 0.24 h. Themixture was concentrated and the residue purified by chromatography(silica, 10% diethyl ether/isohexane as eluent) to give the sub-titlecompound (3.30 g, 84%) as a yellow solid.

¹H NMR 300 MHz (CDCl₃) 7.95 (2H, m), 7.56 (1H, m), 7.28–7.44 (7H, m),4.94 (1H, m), 4.75 (1H, bs), 3.70 (2H, m), 1.38 (9H, s).

c) 1,1-Dimethylethyl[(2S)-2-[(3-cyano-6-methyl-2-pyridinyl)thio]-2-phenylethyl]carbamate

To a mixture of 2-chloro-6-methyl-3-pyridinecarbonitrile (107 mg) andthe product from step (b) (250 mg) in methanol (10 ml) and water (1 ml)was added potassium carbonate (200 mg). The mixture was stirred at roomtemperature under nitrogen for 20 h. The mixture was then concentrated,water added and extracted with ethyl acetate three times.

The organic layers were combined, washed (MgSO₄), dried (brine) andconcentrated. The residue was purified by chromatography (silica, 20%ethyl acetate/isohexane as eluent) to give the sub-title compound (48mg, 19%) as an oil.

MS APCI+ve^(m)/z 370 ([M+H]⁺).

d) 2-[[(1S)-2-Amino-1-phenylethyl]thio]-6-methyl-3-pyridinecarbonitrilehydrochloride

The product from step (c) (45 mg) was dissolved in 4M HCl in dioxane (5ml). The resulting solution was stirred at room temperature for 15minutes, then the solvent was evaporated off. The residue wasrecrystallised from diethyl ether/ethanol to give the title compound asa white solid (19 mg).

MS APCI+ve^(m)/z 270 ([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 8.17 (2H, bs)8.12 (1H, d), 7.55 (2H, m), 7.38 (3H, m), 7.24 (1H, d), 5.41 (1H, t),3.51 (2H, m), 2.61 (3H, s).

EXAMPLE 3 2-[(2,5-Dichlorophenyl)thio]-2-phenylethylamine hydrochloride

a) 1,1-Dimethylethyl [2-hydroxy-2-phenylethyl]carbamate

Racemic α-(aminomethyl)benzenemethanol (13.7 g) andbis(1,1-dimethylethyl)carbonate (21.8 g) were dissolved in methanol (200ml) and 10% aqueous sodium carbonate (200 ml) added. The mixture wasstirred at room temperature for 24 h, poured into 2M hydrochloric acidand extracted with ethyl acetate. Evaporation of the extract andtrituration with ethyl acetate/hexane gave the sub-title compound as apale yellow solid (27.6 g).

¹H NMR 300 MHz (CDCl₃) 7.27–7.38 (5H, m), 4.92 (1H, m), 4.83 (1H, m),3.50 (1H, m), 3.25 (1H, m), 1.45 (9H, s).

b) 1,1-Dimethylethyl[2-[(2,5-dichlorophenyl)thio]-2-phenylethyl]carbamate

To a solution of triphenylphosphine (1.97 g), 2,5-dichlorothiophenol(0.90 g) and the product from step (a) (1.48 g) in THF (30 ml) undernitrogen at 0° C. was added diethylazodicarboxylate (1.18 ml) dropwiseand the mixture was stirred at 0° C. for 45 minutes, then at roomtemperature for 72 h. The mixture was concentrated and the residuepurified by chromatography (silica, 10% ethyl acetate/isohexane aseluent) to give the sub-title compound as a clear oil (500 mg).

¹H NMR 300 MHz (CDCl₃) 7.28 (7H, m), 7.09 (1H, dd), 4.77 (1H, bs), 4.50(1H, bt), 3.53 (2H, m), 1.43 (9H, s).

c) 2-[(2,5-Dichlorophenyl)thio]-2-phenylethylamine hydrochloride

The product from step (b) (0.4 g) was dissolved in 4M HCl in dioxane (5ml). The resulting solution was stirred at room temperature for 6 h, thesolvent was evaporated and the residue triturated with ethyl acetate togive of the title compound (0.24 g) as, a white solid.

MS APCI+ve^(m)/z 298/300 ([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 8.25 (3H,bs), 7.62 (1H, d), 7.55–7.26 (7H, m), 4.97 (1H, m), 3.45 (1H, m), 3.28(1H, m).

EXAMPLE 4 2-[[(1S)-2-Amino-1-phenylethyl]thio]-4-chlorobenzonitrilehydrochloride

a) 1,1-Dimethylethyl[(2S)-2-[(5-chloro-2-cyanophenyl)thio]-2-phenylethyl]carbamate

To a solution of the product from Example 2 step (b) (360 mg) in ethanol(20 ml) was added a solution of sodium hydroxide (90 mg) in water (5ml). After stirring for 5 minutes, 4-chloro-2-fluorobenzonitrile (156mg) was added. The mixture was stirred at room temperature undernitrogen for 24 h. The mixture was then concentrated, water added andextracted with ethyl acetate three times. The organic layers werecombined, washed (MgSO₄), dried and concentrated. The residue waspurified by chromatography (silica, 10% diethyl ether/isohexane aseluent) to give the sub-title compound (60 mg) as an oil.

MS APCI+ve^(m)/z 289 ([M-Boc+H]⁺). ¹H NMR 300 MHz (CDCl₃) 7.50 (1H, d),7.43 (1H, s), 7.37–7.21 (6H, m), 4.82 (1H, bs), 4.60 (1H, t), 3.60 (2H,m), 1.42 (9H, s).

b) 2-[[(1S)-2-Amino-1-phenylethyl]thio]-4-chlorobenzonitrilehydrochloride

The title compound (34 mg) was prepared by the method of Example 2 step(d) using the product from step (a) above.

MS APCI+ve^(m)/z 289 ([M+H]⁺).

¹H NMR 300 MHz (DMSO-d₆) 8.18 (3H, bs), 7.88 (1H, d), 7.84 (1H, m), 7.57(1H, dd), 7.46–7.32 (5H, m), 4.98 (1H, t), 3.51 (1H, dd), 3.33 (1H, m).

EXAMPLE 5N-[2-(5-Chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-ethanimidamidehydrochloride

The title compound was prepared by treating the product of Example 1step (c) (0.2 g) in dry THF (10 ml) with methyl acetimidatehydrochloride (75 mg) followed by triethylamine (0.1 ml) and stirringovernight. The reaction mixture was poured into water and extracted withethyl acetate. The extract was evaporated and the residue purified bychromatography (silica, 2–20% 7M ammonia in methanol/dichloromethane aseluent) to give a glassy solid. The solid was dissolved in methanol (10ml), treated with 1M HCl in ether (1 ml) then evaporated. The residuewas triturated with ether to give the title compound as a cream solid(90 mg). M.p. 133–135° C.

MS APCI+ve^(m)/z 332/334 ([M+H]⁺). ¹H NMR 400 MHz (DMSO-d₆) 9.79 (1H,bt), 9.53 (1H, bs), 8.8 (1H, bs), 8.04 (1H, d), 7.45 (6H, m), 5.84 (1H,m). 3.87 (1H, m), 3.68 (1H, m), 2.18 (3H, s).

EXAMPLE 6N-[2-(5-Chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-2-hydroxyethanimidamidehemi ethanedioate

The title compound was prepared by the method of Example 5 using ethyl2-hydroxyethanimidate hydrochloride in metianol and the ethanedioic acidsalt of the product from Example 1 step (c). M.p. 196° C.

MS APCI+ve^(m)/z 348 ([M+H]⁺). ¹H NMR 400 MHz (DMSO-d₆) 8.0 (1H, dt),7.62–7.33 (6H, in), 5.84 (1H, dd), 4.17 (2H, s), 3.8 (1H, dd), 3.56 (1H,dd).

EXAMPLE 72-[[(1S)-2-Amino-1-phenylethyl]thio]-6-(trifluoromethyl)-3-piridinecarbonitrile

a) 1,1-Dimethylethyl[(2S)-2-[[3-cyano-6-(trifluoromethyl)-2-pyridinyl]thio]-2-phenylethyl]-carbamate

The sub-title compound (115 mg) was prepared by the method of Example 4(a) using the product from Example 2 step (b) and2-chloro-6-(trifluoromethyl)-3-pyridinecarbonitrile.

MS APCI+ve^(m)/z 324 ([M-Boc+H]⁺). ¹H NMR 300 MHz (CDCl₃) 7.94 (1H, d),7.46–7.26 (6H, m), 5.23 (1H, t), 4.79 (1H, bs), 3.75 (2H, m), 1.39 (9H,s).

b)2-[[(1S)-2-Amino-1-phenylethyl]thio]-6-(trifluoroimethyl)-3-pyridinecarbonitrileethandioate

The product from step (a) (110 mg) was dissolved in a solution of 4M HClin dioxane (8 ml). The mixture was stirred at room temperature for 2 h,then evaporated to dryness. The residue was dissolved in aqueous sodiumbicarbonate solution and extracted with dichloromethane (three times).The combined extracts were washed with brine, dried (MgSO₄) andevaporated. The residue was dissolved in ethanol and a solution ofoxalic acid in ethanol added and the resultant mixture evaporated todryness, then recrystallised from a mixture of ethanol and diethyl etherto give the title compound (85 mg) as a white solid.

MS APCI+ve^(m)/z 324 ([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 8.56 (1H, d),7.87 (1H, d), 7.55 (2H, d), 7.41-7.30 (3H, m), 5.33 (1H, t), 3.65–3.48(2H, m).

EXAMPLE 8 2-[[(1S)-2-Amino-1-phenylethyl]thio]-4-methoxybenzonitrilehydrochloride

a) 1,1-Dimethylethyl[(2S)-2-[2-cyano-5-methoxyphenyl)thio]-2-phenylethyl]-carbamate

The product from Example 2 step (b) (360 mg) was stirred in 7M ammoniain methanol (10 ml) for 3 h. The solvent was evaporated, the residuedissolved in dry DMF (10 ml) under nitrogen and2-fluoro-4-methoxybenzonitrile (155 mg) added followed by caesiumcarbonate (655 mg). The reaction mixture was stirred under nitrogen atroom temperature for 3 days then poured into water and extracted withethyl acetate. The combined organic layers were washed with brine anddried (MgSO₄). The solvent was evaporated and the residue purified bychromatography (silica, 10% ethyl acetate/isohexane as eluent) to givethe sub-title compound (115 mg) as an oil.

MS APCI+ve^(m)/z 285 ([M-Boc+H]⁺). ¹H NMR 300 MHz (CDCl₃) 7.50 (1H, d),7.30 (5H, m), 6.92 (1H, s), 6.75 (1H, d), 5.30 (1H, bs), 4.59 (1H, t),3.76 (3H, s), 3.61 (2H, m), 1.41 (9H, s).

b) 2-[[(1S)-2-Amino-1-phenylethyl]thio]-4-methoxybenzonitrilehydrochloride

The title compound (65 mg) was prepared by the method of Example 2 step(d) using the product from step (a) above.

MS APCI+ve^(m)/z 285 ([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 8.24 (3H, bs),7.77 (1H, d), 7.46–7.32 (5H, m), 7.22 (1H, d), 7.01 (1H, dd), 5.01 (1H,t), 3.86 (3H, s), 3.47 (1H, m), 3.28 (1H, m).

EXAMPLE 94-Chloro-2-[[(1S)-2-(methylamino)-1-phenylethyl]thio]benzonitrilehydrochloride

a) 1,1-Dimethylethyl[(2S)-2-[(5-chloro-2-cyanophenyl)thio]-2-phenylethyl]methylcarbamate

To a solution of the product from Example 4 step (a) (92 mg) in THF (5ml) was added sodium hydride (60% in oil; 10 mg) followed by iodomethane(60 μl). The mixture was stirred at room temperature for 2 days thenpoured into water and extracted with diethyl ether (three times). Thecombined organic layers were washed with brine and dried (MgSO₄). Thesolvent was evaporated off to give the sub-title compound (100 mg) as anoil.

MS APCI+ve^(m)/z 303 ([M-Boc+H]⁺).

b) 4-Chloro-2-[[(1S)-2-(methylamino)-1-Rhenylethyl]thio]benzonitrilehydrochloride

The title compound (30 mg) was prepared by the method of Example 2 step(d) using the product from step (a) above.

MS APCI+ve^(m)/z 303/5([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 7.85 (2H, m),7.56 (1H, dd), 7.47–7.33 (5H, m), 5.10 (1H, t), 3.65 (1H, m), 3.48 (1H,m), 2.56 (3H, s).

EXAMPLE 102-[[(1R)-2-Amino-1-(3-isoxazolyl)ethyl]oxy]-4-chloro-5-fluorobenzonitrileethanedioate

a) (R)-2-Hydroxy-2-(3-isoxazolyl)ethylamine

To ytterbium(III) chloride in acetonitrile (10 ml) was added2,6-bis[(4S)-(−)-isopropyl-2-oxazolin-2-yl]pyridine (180 mg) and thesolution stirred for 1 h. After cooling to 0° C.,3-isoxazolecarboxaldehyde (580 mg) in acetonitrile (5 ml) followed byTMSCN (0.96 ml) were added. After warming to room temperature overnight,aqueous ammonium chloride solution was added and the product extractedinto diethyl ether. The extracts were dried (Na₂SO₄), the solvent wasevaporated off, and the residue taken up in diethyl ether (50 ml). Aftercooling to 0° C., the solution was added to lithium aluimnium hydride(570 mg) in diethyl ether (10 ml) at 0° C. After 3 h at roomtemperature, water (0.54 ml), 15% aqueous sodium hydroxide (1.62 ml) andwater (0.54 ml) were added sequentially. The mixture was filteredthrough celite and the solvent was evaporated off to give the sub-titlecompound as a yellow oil (250 mg, 40% ee).

¹H NMR 400 MHz (CDCl₃) 8.38 (1H, d), 6.43 (1H, d), 4.82 (1H, t), 3.14(1H, dd), 3.09 (1H, dd).

b)2-[[(1R)-2-Amino-1-(3-isoxazolyl)ethyl]oxy]-4-chloro-5-fluorobenzonitrileethanedioate

The product from step (a) (8.0 mg) in DMF (2 ml) was treated with 60%sodium hydride in mineral oil (75 mg) with stirring under nitrogen.After 30 minutes, solid 4-chloro-2,5-difluorobenzonitrile (131 mg) wasadded, and the reaction mixture stirred for 16 h, poured into water, andextracted with diethyl ether. The extract was washed with a mixture ofbrine and 1N sodium hydroxide solution (1:1) and then dried (Na₂SO₄).The solvent was evaporated off and the residue purified by passagethrough SCX resin (0–7M ammonia in methanol). The ethandioate salt wasformed in methanol to give the title compound (6 mg) as a brown solid.

¹H NMR 300 MHz (CD₃OD) 8.79 (1H, s), 7.72 (1H, d), 7.52 (1H, d), 6.69(1H, s), 6.01 (1H, dd), 3.77 (1H, dd), 3.60 (1H, dd).

EXAMPLE 11 2-[[2-Amino-1-(3-pyridinyl)ethyl]thio]-4-chlorobenzonitrile

a) 1,1-Dimethylethyl N-[2-(methoxymethylamino)-2-oxoethyl]carbamate

N,O-Dimethylhydroxylamine hydrochloride (1.95 g), EDCI (3.83 g),N-methylmorpholine (2.2 ml) and DMAP (2.44 g) were added to a solutionof N-[(1,1-dimethylethoxy)carbonyl]glycine (3.5 g) in dichloromethane(60 ml) and then stirred at room temperature for 3 days. Dichloromethane(100 ml) was added and then the solution was washed with 2M hydrochloricacid (three times), NaHCO₃ solution (three times) and water (threetimes) and then dried (MgSO₄). The organic layer was separated and theaqueous layer was further extracted twice. The solvent was evaporated togive the sub-title compound (3.42 g).

¹H NMR 300 MHz (CDCl₃) 5.22 (1H, bs), 4.09 (2H, d), 3.72 (3H, s), 3.46(9H, s), 3.21 (3H, s).

b) 1,1-Dimethylethyl [2-oxo-2-(3-pyridinyl)ethyl]carbamate

Isopropylmagnesium bromide (15.5 ml, 2M in THF) was added to a solutionof 3-bromopyridine (4.92 g) in THF (30 ml) at 0° C. and stirred for 1 h.A solution of the product from step (a) (2.18 g) in THF (20 ml) was thenadded and the reaction mixture was stirred at room temperature for 40 h.The mixture was quenched by the addition of saturated ammonium chloridesolution (250 ml) and extracted with ethyl acetate. The organic layerwas washed with brine and dried (MgSO₄). The solvent was evaporated offand the residue purified by chromatography (silica, 20% ethylacetate/dichloromuethane as eluent) to give the sub-title compound (1.84g) as a white solid.

MS APCI+ve^(m)/z 237 ([M+H]⁺). ¹H NMR 300 MHz (CDCl₃) 9.18 (1H, d), 8.83(1H, q), 8.24 (1H, dd), 7.46 (1H, dd), 5.47 (1H, bs), 4.67 (2H, d), 1.48(9H, s).

c) 1,1-Dimethylethyl [2-hydroxy-2-(3-pyridinyl)ethyl]carbamate

To a solution of the product from step (b) (1.30 g) in ethanol (50 ml)was added sodium borohydride (105 mg). The mixture was stirred for 1.5 hthen evaporated. Water was added and the mixture extracted twice withethyl acetate. The combined organic layers were washed (brine) and dried(MgSO₄). The solvent was evaporated off to give the sub-title compound(1.35 g) as an oil.

MS APCI+ve^(m)/z 239 ([M+H]⁺). ¹H NMR 300 MHz (CDCl₃) 8.59 (1H, m), 8.53(1H, m), 7.73 (1H, d), 7.30 (1H, m), 4.97 (1H, bs), 4.90 (1H, m), 3.75(1H, bs), 3.50 (1H, m), 3.30 (1H, m), 1.45 (9H, s).

d)S-[2-[[(1,1-Dimethylethoxecarbonyl]amino]-1-(3-pyridinyl)ethyl]benzenecarbothioate

The sub-title compound (740 mg) was prepared by the method of Example 2step (b) using the product from step (c) above.

MS APCI+ve^(m)/z 359([M+H]⁺). ¹H NMR 300 MHz (CDCl₃) 8.67 (1H, d), 8.53(1H, d), 7.94 (2H, d), 7.74 (1H, d), 7.57 (1H, t), 7.45 (2H, t), 7.27(1H, m), 4.95 (1H, t), 4.79 (1H, bs), 3.71 (2H, t), 1.38 (9H, s).

e) 1,1-Dimethylethyl[2-[(5-chloro-2-cyanophenyl)thio]-2-(3-pyridinyl)ethl]carbamate

The sub-title compound (300 mg) was prepared by the method of Example 8step (a) using oa the product from step (d) above.

MS APCI+ve^(m)/z 390/2 ([M+H]⁺). ¹H NMR 300 MHz (CDCl₃) 8.52 (2H, m),7.73 (1H, d), 7.53 (1H, d), 7.45 (1H, s), 7.30 (2H, m), 4.85 (1H, bs),4.63 (1H, t), 3.62 (2H, m), 1.41 (9H, s).

f) 2-[[2-Amino-1-(3-piyridinyl)ethyl]thio]-4-chlorobenzonitriledihydrochloride

The title compound (212 mg) was prepared by the method of Example 2 step(d) using the product from step (e) above.

MS APCI+ve^(m)/z 290/2 ([M+H]⁺). ¹H NMR 300 MHz (DMSO-d₆) 8.89 (1H, s),8.76 (1H, d), 8.43 (3H, bs), 8.31 (1H, d), 8.04 (1H, s), 7.90 (1H, d),7.80 (1H, t), 7.64 (1H, d), 5.18 (1H, t), 3.65 (1H, m), 3.46 (1H, m).

Screens

The pharmacological activity of compounds according to the invention wastested in the following screens.

Screen 1

The activity of compounds of formula (I), or a pharmaceuticallyacceptable salt thereof, may be screened for nitric oxide synthaseinhibiting activity by a procedure based on that of Förstermann et al.,Eur. J. Pharm., 1992, 225, 161–165. Nitric oxide synthase converts³H-L-arginine into ³H-L-citrulline which can be separated by cationexchange chromatography and quantified by liquid scintillation counting.

Enzyme is prepared, after induction, from the cultured murine macrophagecell line J774A-1 (obtained from the laboratories of the Imperial CancerResearch Fund). J774A-1 cells are cultured in Dulbeccos Modified EaglesMedium (DMEM) supplemented with 10% foetal bovine serum, 4 mML-glutamine and antibiotics (100 units/nl penicillin G, 100 mg/mlstreptomycin & 0.25 mg/ml ainphotericin B). Cells are routinely grown in225 cm³ flasks containing 35 ml medium kept at 37° C. and in ahumidified atmosphere containing 5% CO₂.

Nitric oxide synthase is produced by cells in response to interferon-g(IFNg) and lipopolysaccharide (LPS). The medium from confluent cultureflasks is removed and replaced with 25 ml (per flask) of fresh mediumcontaining 1 mg/ml LPS and 10 units/ml IFNg. After a period of 17–20hours in culture, harvesting of cells is accomplished by scraping thecell sheet from the flask surface into the culture medium. Cells arecollected by centrifugation (1000 g for 10 minutes) and lysate preparedby adding to the cell pellet a solution containing 50 mM Tris-HCl (pH7.5 at 20° C.), 10% (v/v) glycerol, 0.1% (v/v) Triton-X-100, 0.1 mMdithiothreitol and a cocktail of protease inhibitors comprisingleupeptin (2 mg/ml), soya bean trypsin inhibitor (10 mg/ml), aprotinin(5 mg/ml) and phenylmethylsulphonyl fluoride (50 mg/ml).

For the assay, 25 μl of substrate cocktail (50 mM Tris-HCl (H 7.5 at 20°C.), 400 μM NADPH, 20 μM flavin adenine dinucleotide, 20 μM flavinmononucleotide, 4 μM tetrahydrobiopterin, 12 μM L-arginine and 0.025mCiL-[³H] arginine) is added to wells of a 96 well filter plate (0.45 μMpore size) containing 25 μl of a solution of test compound in 50 mMTris-HCl. The reaction is started by adding 50 μl of cell lysate(prepared as above) and after incubation for 1 hour at room temperatureis terminated by addition of 50 μl of an aqueous solution of 3 mMnitroarginine and 21 mM EDTA.

Labelled L-citrulline is separated from labelled L-arginine using DowexAG-50W. 150 μl of a 25% aqueous slurry of Dowex 50W (Na⁺ form) is addedto the assay after which the whole is filtered into 96 well plates. 75μl of filtrate is sampled and added to wells of 96 well platescontaining solid scintillant. After allowing the samples to dry theL-citrulline is quantified by scintillation counting.

In a typical experiment basal activity is 300 dpm per 75 μl sample whichis increased to 1900 dpm in the reagent controls. Compound activity isexpressed as IC₅₀ (the concentration of drug substance which gives 50%enzyme inhibition in the assay) and ainlinoguanidine, which gives anIC₅₀ (50% inhibitory concentration) of 10 μM, is tested as a standard toverify the procedure. Compounds are tested at a range of concentrationsand from the inhibitions is obtained IC₅₀ values are calculated.Compounds that inhibit the enzyme by at least 25% at 100 μM are classedas being active and are subjected to at least one retest.

In the above screen, the compounds of Examples 1 to 11 were tested andgave IC₅₀ values of less than 10 μM indicating that they are expected toshow useful therapeutic activity.

Screen 2

Recombinant human NO synthases (iNOS, eNOS & nNOS) were expressed in E.coli and lysates were prepared in Hepes buffer (pH 7.4) containingco-factors (FAD, FMN, H₄B), protease inhibitors, lysozyme and thedetergent, CHAPS. These preparations were used, at suitable dilution, toassess inhibition of the various isoforms. Inhibition of NOS wasdetermined by measuring the formation of L-[³H]citrulline fromL-[³H]argirine using an adaptation of the method of Försterrnann et al.⁹Enzyme assays were performed in the presence of 3 μM [³H]arginine, 1 mMNADPH and other co-factors required to support NOS activity (FAD, FMN,H₄B, calmodulin, Ca²⁺). Since various NOS irhibitors have been reportedto exhibit slow binding kinetics, or to inactivate the enzyme in a timedependent manner, enzyme and inhibitor were pre-incubated for 60 min inthe presence of NADPH before addition of arginine to initiate thereaction. Incubations continued for a further 60 min before the assayswere quenched and [³H]citrulline separated from unreacted substrate bychromatography on Dowex-50W resin in a 96-well format.

In the above screen, the compounds of Examples 1 to 11 were tested andgave IC₅₀ values of less than 10 μM against the iNOS enzyme indicatingthat they are expected to show useful therapeutic activity.

Screen 3

Compounds also show activity against the human form of induced nitricoxide synthase as can be demonstrated in the following assay.

The human colorectal carcinoma cell line, DLD-1 (obtained from theEuropean Collection of Animal Cell Culture—cell line number 90102540)was routinely grown in RPMI 1640 supplemented with 10%(v/v) foetalbovine serum, and 2 mM L-glutanmine, at 37° C. in 5% CO₂.

Nitric oxide synthase was induced in cells by addition of mediumcontaining human recombinant gamma-IFN (1000 units/ml), TNF-alpha (200U/ml), IL-6 (200 U/ml) and IL-1-beta (250 U/ml). After incubation for 18hours at 37° C., the medium was removed and the cells washed with warmphosphate buffered saline. Cells were incubated for a further 5 hours at37° C./5% CO₂ in RPMI 1640 containing 100 μM L-arginine and 100 μMverapamil-HCl in the presence and absence of test compounds.

Nitrite accumulation was determined by mixing an equal volume of culturemedia with Griess reagent (10 mg/ml sulphanilamide, 1 mgN-(1-naphthyl)ethylenediamine in 1 ml 2.5% (v/v) phosphoric acid).Inhibition in the presence of compounds was calculated relative to thenitrite levels produced by untreated cells. IC₅₀ values were estimatedfrom a semi-log plot of % inhibition versus concentration of compound.

In this screen the compounds of Examples 1 to 11 gave IC₅₀ values ofless than 100 μM, indicating that they are predicted to show usefultherapeutic activity.

1. A compound of formula (I)

wherein: X represents H, C1 to 4 alkyl, C1 to 4 alkoxy, halogen, OH, CN,C≡CH, NO₂, CHO, COCH₃ or NHCHO; said alkyl or alkoxy group beingoptionally further substituted by one or more fluorine atoms or by an OHgroup; Y represents C1 to 4 alkyl, C1 to 4 alkoxy, halogen, OH, CN,C≡CH, NO₂, CHO, COCH₃ or NHCHO; said alkyl or alkoxy group beingoptionally further substituted by one or more fluorine atoms; Either oneof T, U and W represents N and the other two independently representCR⁴; or each of T, U and W represents CR⁴; and each R⁴ groupindependently represents H, F or CH₃; V represents O or S(O)_(n); nrepresents an integer 0, 1 or 2; R¹ represents C2 to 4 alkenyl, C2 to 4alkynyl, C3 to 6 cycloalkyl or a 4 to 8 membered saturated heterocyclicring incorporating one heteroatom selected from O, S and N; any of saidgroups being optionally further substituted by C1 to 4 alkyl, C1 to 4alkoxy, C1 to 4 alkylthio, C3 to 6 cycloalkyl, one or more halogens orphenyl; said phenyl group being optionally further substituted by one ormore substituents selected independently from halogen, C1 to 4 alkyl, C1to 4 alkoxy, CF₃, OCF₃, CN or NO₂; or R¹ represents phenyl or a five orsix membered aromatic heterocyclic ring containing 1 to 3 heteroatomsindependently selected from O, S and N; said phenyl or aromaticheterocyclic ring being optionally substituted by one or moresubstituents selected independently from halogen, C1 to 4 alkyl, C1 to 4alkoxy, OH, CN, NO₂ or NR⁵R⁶; said alkyl or alkoxy group beingoptionally further substituted by one or more fluorine atoms; R² and R³independently represent H, C1 to 4 alkyl or C3 to 6 cycloalkyl; saidalkyl group being optionally substituted by one or more substituentsselected independently from C1 to 4 alkoxy, halogen, OH, NR⁷R⁸, ═NR⁷,phenyl or a five or six membered aromatic or saturated heterocyclic ringcontaining 1 to 3 heteroatoms independently selected from O, S and N;said phenyl or aromatic heterocyclic ring being optionally furthersubstituted by halogen, C1 to 4 alkyl, C1 to 4 alkoxy, CF₃, OCF₃, CN orNO₂; and R⁵, R⁶, R⁷ and R⁸ independently represent H or C1 to 4 alkyl;or a pharmaceutically acceptable salt thereof.
 2. A compound of formula(I), according to claim 1, wherein V represents O.
 3. A compound offormula (I), according to claim 1, wherein V represents S(O)_(n) and nrepresents O.
 4. A compound of formula (I), according to claim 1,wherein X and Y independently represent Br, Cl, CH₃, CH₂F, CHF₂, CF₃ orCN.
 5. A compound according to claim 4 wherein Y represents CN.
 6. Acompound of formula (I), according to claim 1, which is:2-{[(1S)-2-amino-1-phenylethyl]oxy}-4-chloro-5-fluorobenzonitrile;2-[[(1S)-2-amino-1-phenylethyl]thio]-6-methyl-3-pyridinecarbonitrile;2-[(2,5-dichlorophenyl)thio]-2-phenylethylamine; or a pharmaceuticallyacceptable salt thereof.
 7. A pharmaceutical composition comprising acompound of formula (I) according to claim 1, or a pharmaceuticallyacceptable salt thereof, in admixture with a pharmaceutically acceptableadjuvant, diluent or carrier.
 8. A method of treating, inflammatorydisease in a person in need thereof, wherein the method comprisesadministering to the person a therapeutically effective amount of acompound of formula (I), as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 9. A method of treating, CNS disease in aperson in need thereof, wherein the method comprises administering tothe person a therapeutically effective amount of a compound of formula(I) as defined in claim 1, or a pharmaceutically acceptable saltthereof.
 10. A process for the preparation of a compound of formula (I),as defined in claim 1, or a pharmaceutically acceptable salt, enantiomeror racemate thereof, wherein the process comprises: (a) reaction of acompound of formula (II)

 wherein T, U, X, Y and W are as defined in claim 1 and L represents aleaving group, with a compound of formula (III)

 wherein R¹, R², R³ and V are as defined in claim 1; or (b) reaction ofa compound of formula (TV)

 wherein T, U, W, X, Y and V are as defined in claim 1, with a compoundof formula (V)

 wherein R¹, R² and R³ are as defined in claim 1 and L is a leavinggroup; or (c) reaction of a compound of formula (VI)

 wherein R¹, T, U, W, X, Y and V are as defined in claim 1 and L³ is aleaving group, with a compound of formula (VII)R²R³NH  (VII)  wherein R² and R³ are as defined in claim 1; or (d)reduction of a compound of formula (VIII)

 wherein R¹, T, U, W, X, Y and V are as defined in claim 1 and Qrepresents azide (N₃); or (e) hydrolysis of a compound of formula (VIII)

 wherein T, U, W, X, Y and V are as defined in claim 1 and Q representsan imide group; and where desired or necessary converting the resultantcompound of formula (I), or another salt thereof, into apharmaceutically acceptable salt thereof; or converting one compound offormula (I) into another compound of formula (I); and where desiredconverting the resultant compound of formula (I) into an optical isomerthereof.
 11. A method of treating, inflammatory bowel disease in aperson in need thereof wherein the method comprises administering to theperson a therapeutically effective amount of a compound of formula (I),as defined in claim 1, or a pharmaceutically acceptable salt thereof.12. A method of treating, rheumatoid arthritis in a person in needthereof wherein the method comprises administering to the person atherapeutically effective amount of a compound of formula (I), asdefined in claim 1, or a pharmaceutically acceptable salt thereof.
 13. Amethod of treating, osteoarthritis in a person in need thereof, whereinthe method comprises administering to the person a therapeuticallyeffective amount of a compound of formula (I), as defined in claim 1, ora pharmaceutically acceptable salt thereof.
 14. A method for treatingpain in a person in need thereof wherein the method comprisesadministering to the person a therapeutically effective amount of acompound of formula (I), as defined in claim 1, or a pharmaceuticallyacceptable salt thereof.
 15. A method of treating, migraine in a personin need thereof, wherein the method comprises administering to theperson a therapeutically effective amount of a compound of formula (I),as defined in claim 1, or a pharmaceutically acceptable salt thereof.16. A composition comprising a compound of claim 1, in combination witha COX-2 inhibitor.
 17. A method of treating an inflammatory disease, themethod comprising administering to a person in need thereof atherapeutically effective amount of the composition of claim
 16. 18. Thecompound of claim 1, wherein R¹ represents phenyl or a five or sixmembered aromatic heterocyclic ring containing 1 to 3 heteroatomsindependently selected from O, S and N; said phenyl or aromaticheterocyclic ring being optionally substituted by one or moresubstituents selected independently from halogen, C1 to 4 alkyl, C1 to 4alkoxy, OH, CN, NO₂ or NR⁵R⁶; said alkyl or alkoxy group beingoptionally further substituted by one or more fluorine atoms.
 19. Thecompound of claim 1, wherein the compound is2-[[(1S)-2-amino-1-phenylethyl]thio]-4-chlorobenzonitrile;N-[2-(5-chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-ethanimidamide;N-[2-(5-chloro-2-cyano-4-fluorophenoxy)-2-phenylethyl]-2-hydroxyethanimidamide;2-[[(1S)-2-amino-1-phenylethyl]thio]-6-(trifluoromethyl)-3-pyridinecarbonitrile;2-[[(1S)-2-amino-1-phenylethyl]thio]-4-methoxybenzonitrile;4-chloro-2-[[(1 S)-2-(methylamino)-1-phenylethyl]thio]benzonitrile;2-[[(1R)-2-amino-1-(3-isoxazolyl)ethyl]oxy]-4-chloro-5-fluorobenzonitrile;2-[[2-amino-1-(3-pyridinyl)ethyl]thio]-4-chlorobenzonitrile; or apharmaceutically acceptable salt thereof.
 20. A pharmaceuticalcomposition comprising a compound of formula (I) according to claim 18,or a pharmaceutically acceptable salt thereof, in admixture with apharmaceutically acceptable adjuvant, diluent or carrier.
 21. Apharmaceutical composition comprising a compound of formula (I)according to claim 19 , or a pharmaceutically acceptable salt thereof,in admixture with a pharmaceutically acceptable adjuvant, diluent orcarrier.